The overall goal of this project is to understand the catalytic mechanisms of three reactions that are promoted within the ribosome: peptide bond formation and peptide release within the 50S peptidyl transferase center (PTC) and mRNA cleavage within the 30S decoding center. Peptide Bond Formation and Peptide Release (Aims 1 and 2). During protein synthesis, the ribosome catalyzes peptide bond formation (aminolysis) during amino acid polymerization and peptide release (hydrolysis) after the last peptide linkage is made. Understanding how the ribosome catalyzes these two competing and essential reactions has been a long-standing biochemical goal; yet fundamental questions remain unanswered. Does the ribosome strictly provide entropic stabilization by aligning the nucleophiles ?-amino group or water) or does it contribute chemically to catalysis? How do the transition states of the catalyzed and uncatalyzed reactions compare? Is deprotonation of the amine and protonation of the leaving group concerted or stepwise? Do both reactions proceed through a tetrahedral intermediate? What contribution is made by functional groups in the PTC, the tRNA substrate and the release factor? Addressing these questions requires detailed understanding of the reaction transition state (TS) and the importance of potential stabilizing interactions within the ribosomal active site. A series of complementary, yet fundamentally different experimental approaches will reveal the mechanism of these biologically essential reactions. These approaches include: i. isotopically labeled substrates to define the rate-limiting bond breaking and bond forming steps (isotope exchange and kinetic isotope effect analysis, KIE) ii. pKa perturbed substrates to establish the charge distribution in the transition state (Bronsted analysis); and iii. Novel analogs to define the orientation of the water and the contribution made by charge stabilization and hydrogen bonding to the peptide released TS. mRNA Cleavage by RelE (Aim 3). mRNA is cleaved by RelE during the bacterial stringent response, but RelE only cuts translating mRNAs bound to the ribosome. The activity of RelE and related toxins may allow fast adaptation of bacterial cells to environmental changes through global modulation of their translation rate. Although RelE is structurally similar to the general family of endoribonucleases, it does not contain any of the residues expected to be important for catalysis. A recent crystal structure of RelE bound to the 70S ribosome established which residues of RelE and the ribosome are near the cleavage site, but the biochemical data did not correlate well with the structural predictions. Complementary biochemical and genetic approaches will be used to understand the mechanism of RelE based cleavage, the nature of the RelE interaction with the ribosome and to test how the ribosome contributes to the cleavage reaction.